Sintilimab with two cycles of chemotherapy for the treatment of advanced squamous non-small cell lung cancer: a phase 2 clinical trial

This was a single-arm, multicenter phase 2 clinical trial (ChiCTR1900021726) involving advanced squamous non-small cell lung cancer (sq-NSCLC) patients undergoing 2 cycles of nab-paclitaxel/carboplatin and sintilimab (anti-PD-1), followed by sintilimab maintenance therapy. The median progression-free survival (PFS) was 11.4 months (95% CI: 6.7-18.1), which met the pre-specified primary endpoint. Secondary endpoints included objective response rate reaching 70.5% and a disease control rate of 93.2%, with a median duration of response of 13.6 months [95% CI: 7.0–not evaluable (NE)]. The median overall survival was 27.2 months (95% CI: 20.2–NE) with treatment-related adverse events grades ≥3 occurring in 10.9% of patients. Predefined exploratory endpoints comprised relationships between biomarkers and treatment efficacy, and the association between circulating tumor DNA (ctDNA) dynamics and PFS. Biomarker analysis revealed that the breast cancer gene 2, BMP/Retinoic Acid Inducible Neural Specific 3, F-box/WD repeat-containing protein 7, tyrosine-protein kinase KIT and retinoblastoma 1 abnormalities led to shorter PFS, while ctDNA negative at baseline or clearance at 2 cycles of treatment was associated with longer PFS (18.1 vs. 4.3 months). Taken together, sintilimab in combination with 2 cycles of nab-paclitaxel/carboplatin treatment produced encouraging PFS and better tolerability as first-line treatment for advanced sq-NSCLC.

2. As in previous study, Pembrolizumab (KeyNote-042) can be used as a monotherapy for patients with high PD-L1 expression and has acceptable therapeutic efficacy.However, in this study, the authors attempted to evaluate the efficacy of short-term chemotherapy for NSCLC, and the proportion of patients with high PD-L1 expression should be discussed, rather than only dividing the PD-L1 expression level of patient into expression and nonexpression subgroups according to the existing statistical data.
3. This samples size is too small to identify the efficacy of this strategy for sq-NSCLC in stage IIIB-IV, which were composed of multiple stage.The treatment strategies of stage IIIB-C would not be unified and their prognosis is heterogeneous.Considering that the proportion of stage III patients included in this study exceeds 20%, please carefully describe the reasons why these patients did not undergo radical surgery, neoadjuvant treatment followed by surgery, or PACIFIC regimens.For example, a portion of patients in stage IIIB who received induction immuno-chemotherapy may undergo radical resection to improve survival.4. According to my understanding, the authors may believe that these biomarkers screened by NGS have the same prognostic efficacy, but I strongly recommend that the authors should profoundly discuss the role and efficacy of each biomarker during the baseline sample analysis by ROC curve.After that, tissue staining should be performed where available to determine whether BRCA2, BRINP3, FBXW7, KIT and RB1 are expressed in tumor cells.
5. Considering authors attempted to establish the relationship between the therapeutic efficacy and the dynamic changes of ctDNA during treatment, whether the relationship between the tumor WES and ctDNA should be evaluated.

Minor issues
1.The expression on page 6, line 136 is difficult to understand, please describe it again.Why baseline samples including C1 and C2 samples.
2. The median follow-up time of this study was 15.2 months, and it is inappropriate to discuss the 2-year survival rate in the section of clinical efficacy (Page 5, Line 98).
3. In Supplementary Tables 1 and 2, there are obvious errors in the statistical methods, and in the case of insufficient sample size (n=44), it is not possible to include all variables into the multivariable Cox regression model.4. In Supplementary Fig. 1, please demonstrated the data of patients whose PD-L1 expression undetectable.5. Please describe the primary endpoint in the Abstract and whether the study has met this point.
6. Please describe the secondary endpoints of the study in the Abstract, as well as the exploratory endpoints mentioned in the study protocol.7.For endpoints which was listed in protocol but not mentioned in the manuscript, please explain the reasons.
8. Considering the OS was discussed by authors, the second-line treatment should also be shown in the article.9.The last follow-up time is January 2023, please update the OS and PFS.Timely prognostic information is the most important endpoint in clinical trial report.10.In the Abstract or in the Main Text, the contents that first appear should not be expressed as Abbreviation.

Reviewer #2 (Remarks to the Author): with expertise in lung cancer, therapy
Zhang and colleagues provide results of a single-arm phase 2 multi-center trial conducted in China.47 Patients received two cycles of carboplatinum, nab-paclitaxel and sintilimab for stage III/IV squamous NSCLC, followed by ICI maintenance.The design of the trial is clearly explained, methodology is sound, results are clearly reported and the conclusions drawn are comprehensible.The trial met its primary endpoint (PFS) and demonstrated encouraging efficacy (ORR, DoR) and survival results (PFS, OS).Furthermore, responses were correlated with biomarkers (blood-and tissue-based) and ct-DNA clearance.
The authors should be congratulated for having conducted this trial.ORR, PFS and OS compare very favorably to what has been reported from comparable trials (KN407, 9LA).
There are some points that make the comparability to established regimens difficult.First, a relatively high share of patients with stage III disease was included (25%; 9LA/KN407: stage IV only, Empower-Lung 3 14% (sq only not reported)), and overall, the number of patients included is small.Second, the estimation of the real effect size is difficult due to the singlearm design.Third, RECIST evaluation was done by the local investigators and was not confirmed centrally.And forth, follow-up time is quite short (15 months; only 21 PFS and 18 OS events).Concerning baseline characteristics there is some overweight of male patients, and the share of patients with undocumented PD-L1 expression level is relatively high (>50%).The nab-paclitaxel dose chosen (260 mg/m2 q3w) does not represent an established dose regimen for NSCLC (at least in Europe or the U.S.) and should be commented.Whereas TRAE appear to be low, irAE are currently not reported, which I would recommend.Results for biomarkers confirm the current estimation but do not add substantially new information (ct-DNA clearance is associated with favorable outcome; correlation of gene analysis with outcome is prognostic, but as the trial lacks a comparator arm no conclusions can be drawn with regard to a predictive impact).
To conclude, the present investigation adds evidence for palliative first-line treatment of sq NSCLC and suggests that the number of initial cycles of chemotherapy might be limited to two.But we can not categorize the results with double checkpoint inhibition on the one side (CM9LA) and "classical" chemoimmunotherapy on the other side (KN407).

Reviewer #3 (Remarks to the Author): with expertise in biostatistics, clinical trial study design
This is a open-label single-arm phase 2 clinical trial of sintilimab, a PD-1 inhibitor, with two cycles of chemo for treatment-naive advance squamous NSCLC.The primary endpoint is PFS, with second endpoints including ORR, TTR, DOR, OS, and safety.The manuscript is well written with study design, methods and results.
1.The gender is unbalanced (only 1 female included).It's curious why this happened.
2. The Sintilimab is a PD-1 inhibitor, but only half patients tested for PD-L1 and analysis was stratified by TPS<1% VS >1%, why the other half is ND (not detectable)?Why PD-L1 test is not used in screening for everyone?The authors also perform NGS and cfDNA in a subset of patients, which attempted to contribute the promising biomarkers of survival and therapy.This manuscript seems to be an interesting trial, but I think it is unreasonable to evaluate the effect of first-line treatment with 44 people in stage III-IV, where these various diseases are consisted.We have mentioned these points in the revised Trial Design section of the manuscript.

Reply
It is important to realize that it is our intention to conduct a randomized controlled phase 3 clinical trial in the near future.When the time comes, we will gladly welcome and incorporate your valuable suggestions, particularly the inclusion of an appropriate control group.
2. As in previous study, Pembrolizumab (KeyNote-042) can be used as a monotherapy for patients with high PD-L1 expression and has acceptable therapeutic efficacy.However, in this study, the authors attempted to evaluate the efficacy of short-term chemotherapy for NSCLC, and the proportion of patients with high PD-L1 expression should be discussed, rather than only dividing the PD-L1 expression level of patient into expression and nonexpression subgroups according to the existing statistical data.We have added this information to the revised manuscript.Reply: Thank you for your helpful comments.We first explored the association between PFS and gene mutations, and identified that mutations in BRCA2, BRINP3, FBXW7, KIT or RB1 showed a similar association with poorer PFS (P < 0.05, Supplementary Fig. 3).

Supplementary
However, due to the relatively small sample size of patients with mutated genes, conducting additional stratified analyses may introduce a certain degree of bias.
Therefore, we chose to examine the likelihood of any occurrence among 5 genes (BRCA2, BRINP3, FBXW7, KIT or RB1) and simultaneously assessed the predictive effects on survival rates using ROC curves for each individual gene (BRCA2, BRINP3, FBXW7, KIT or RB1).From Supplementary Fig. 4, it is evident that the predictive effectiveness of the 18-month survival rate is significantly enhanced whenever a mutation occurs in any one of these 5 genes (BRCA2, BRINP3, FBXW7, KIT or RB1), surpassing that of a single gene as an indicator.
In addition, we currently lack a sufficient quantity of tissue samples (some patients have died) for staining and verification in tumor cells, but we will conduct these tests in a further clinical trial in the very near future.
Since the number of patients with each mutation in the above genes was small, we further combined all mutations and observed a shorter PFS time than in those patients without any of these mutations (Fig. 3B).
CI confidence interval, DOR duration of response, HR hazard ratio, m month, ND not detectable, NE not evaluable, ORR objective response rate, OS overall survival, PFS progression free survival.
5. Please describe the primary endpoint in the Abstract and whether the study has met this point.
Reply: The trial reached its primary endpoint of PFS, information which has been added to the revised Abstract.
6. Please describe the secondary endpoints of the study in the Abstract, as well as the exploratory endpoints mentioned in the study protocol.The authors should be congratulated for having conducted this trial.ORR, PFS and OS compare very favorably to what has been reported from comparable trials (KN407, 9LA).

Reply
There are some points that make the comparability to established regimens difficult.
First, a relatively high share of patients with stage III disease was included (25%; 9LA/KN407: stage IV only, Empower-Lung 3 14% (sq only not reported)), and overall, the number of patients included is small.
Reply: In present trial, we enrolled 11 patients at stage III.Before their inclusion, these patients underwent a comprehensive review by a multidisciplinary tumor board.
Except for 1 patient (stage IIIB) who declined radiotherapy, the remaining 10 patients were found to be unsuitable candidates for curative surgery or chest radiotherapy.
The specific reasons for their ineligibility encompassed various conditions have been added to the revised manuscript as Supplementary Table 1.
Subsequently, we also conducted a comparison with a previously conducted phase  And forth, follow-up time is quite short (15 months; only 21 PFS and 18 OS events).These data have also been updated in the revised manuscript.

Reply
Concerning baseline characteristics there is some overweight of male patients, and the share of patients with undocumented PD-L1 expression level is relatively high (>50%).
Reply: Yes, we agree.All the patients we enrolled were squamous cell lung carcinoma cases, which is the predominant lung cancer type in males and especially low in Whereas TRAE appear to be low, irAE are currently not reported, which I would recommend.
Reply: Thank you for the advice.
We have compiled and analyzed the irAEs recorded in the EDC system.The summary of irAEs has been added as Table 4 to the revised manuscript.
Immune-related adverse events (irAEs) of any grade were reported for 14 (30.with pneumonitis and the other with a rash.There were no grade 4 or 5 irAEs. Results for biomarkers confirm the current estimation but do not add substantially new information (ct-DNA clearance is associated with favorable outcome; correlation of gene analysis with outcome is prognostic, but as the trial lacks a comparator arm no conclusions can be drawn with regard to a predictive impact).
To conclude, the present investigation adds evidence for palliative first-line treatment of sq NSCLC and suggests that the number of initial cycles of chemotherapy might be limited to two.Press, 2021).
2. The Sintilimab is a PD-1 inhibitor, but only half patients tested for PD-L1 and analysis was stratified by TPS<1% VS >1%, why the other half is ND (not detectable)?Why PD-L1 test is not used in screening for everyone?
Reply: Due to the small size of biopsy tissue samples in advanced lung squamous cell carcinoma, we could not perform PD-L1 testing on all patients.In response to the issue you raised and Reviewer 1, we have reanalyzed the expression of PD-L1 in all available tissue samples.Currently, there are PD-L1 results for 33 patients (71.7%).

:
We agree and have addressed this concern in the limitations section of the revised manuscript and added 2 extra relevant references.In addition, the small sample size could only give a hint on the relationship between favorable outcomes on PFS with the combination of a reduced administration of conventional chemotherapy with a PD-1 inhibitor, which has been previously been reported for similar first-line sq-NSCLC therapies with extended chemotherapy cycles (Gandhi et al., 2018; Zhou et al., 2021). Zhou, C. et al.Sintilimab Plus Platinum and Gemcitabine as First-Line Treatment for Advanced or Metastatic Squamous NSCLC: Results From a Randomized, Double-Blind, Phase 3 Trial (ORIENT-12).J. Thorac.Oncol.16, 1501-1511 (2021). Gandhi, L. et al.Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer.N. Engl.J. Med.378, 2078-2092 (2018).Major issues 1.As a phase 2 clinical trial, the purpose of this research assesses the efficacy of shortcourse chemotherapeutic regimen for sq-NSCLC in advanced stage.However, the data of prognosis in this single-arm trial could not validate the purpose, because this study lacked a comparison with standard treatment.What is more, considering the different stage and PD-L1 expression level of tumor probably significantly influence the therapeutic efficacy, therefore, the efficacy of short-course therapy cannot be confirmed by comparing with data from both prospective clinical trials or real-world studies were published previously.Reply: Initially we designated solely chemotherapy as the control group when devising the trial.However, it is worth noting that in 2019, the landscape of cancer treatment in China evolved, with chemotherapy now being complemented by immunotherapy as the new standard treatment regime.As a result, standalone chemotherapy is no longer considered the optimal choice of treatment for patients.We therefore adjusted our trial protocol, as documented in the attached file labeled Protocol No CIBI308Y014.Due to considerations related to securing trial funding and the practical limitations on the number of eligible patients, we ultimately proceeded with a single-arm trial design.

:
We appreciate your concern and in response, we have included the expression of PD-L1 in the analyses.Currently, we have acquired PD-L1 expression data for 33patients.Of these, only 3 displayed high PD-L1 expression (PD-L1 ≥ 50%), while 11 exhibited moderate PD-L1 expression (PD-L1 1-49%) and 19 low PD-L1 expression (PD-L1 < 1%).There were no significant differences in PFS or OS times in patients with different PD-L1 expression levels, but significant differences of DORs were found between the groups.

: 7 . 8 .
The secondary endpoints included the objective response rate (ORR) (with partial response (PR) and complete response (CR)), disease control rate (DCR), time to response (TTR), duration of response (DOR), overall survival (OS) and safety indicators.The exploratory endpoints included the relationship between biomarkers in tumor tissue and treatment efficacy, and evaluation of the association between ctDNA in peripheral blood and treatment efficacy.For endpoints which was listed in protocol but not mentioned in the manuscript, please explain the reasons.Reply: In this research, we performed an analysis and reported on the primary trial endpoint, PFS, as well as the secondary trial endpoints (ORR, DCR, TTR, DOR, OS, and safety indicators) mentioned in the protocol.However, due to limitations, such as constrained funding and a scarcity of tissue samples, we were unable to collect the expected data for exploratory endpoints, except for the changes in blood ctDNA during the treatment process and its correlation with efficacy.Considering the OS was discussed by authors, the second-line treatment should also be shown in the article.Reply: We have added the following information to the revised Results section: Up to the cut-off date on August 31 2023, 3 patients completed two years of planned immunotherapy as scheduled.PD was observed in 27 patients (61.4%), and among them, 22 initiated second-line treatments, of whom 14 received chemotherapy (12 with gemcitabine + platinum-based agents, and 2 with paclitaxel + platinum-based agents), 5 received immunotherapy combinations (3 combined with chemotherapy, 2 in combination with anlotinib), and 2 were treated solely with anlotinib, while 1 patient underwent radiotherapy for bone metastatic lesions.The OS for second-line treatment patients was 21.2 months (95% CI: 14.7-32.4months) (Supplementary Fig.1).Zhang and colleagues provide results of a single-arm phase 2 multi-center trial conducted in China.47 Patients received two cycles of carboplatinum, nab-paclitaxel and sintilimab for stage III/IV squamous NSCLC, followed by ICI maintenance.The design of the trial is clearly explained, methodology is sound, results are clearly reported and the conclusions drawn are comprehensible.The trial met its primary endpoint (PFS) and demonstrated encouraging efficacy (ORR, DoR) and survival results (PFS, OS).Furthermore, responses were correlated with biomarkers (blood-and tissue-based) and ct-DNA clearance.
(10.5 cm) involving left main bronchus and causing complete lung collapse 03003 ⅢB A large cystic lesion containing an air-fluid level 06001 ⅢB Recurrent left lung cancer invading the left and right main bronchus, high risk for radiotherapy Second, the estimation of the real effect size is difficult due to the single-arm design.Reply: Yes, we agree, as a single-arm clinical trial, indeed, this is a limitation (we have already added this limitation to the revised manuscript text).We intend to conduct a randomized controlled phase 3 clinical trial in the near future to validate our findings.Initially we designated solely chemotherapy as the control group when devising the trial.However, it's worth noting that in 2019, the landscape of cancer treatment in China evolved, with chemotherapy now being complemented by immunotherapy as the new standard regime.As a result, standalone chemotherapy is no longer considered the optimal choice of treatment for patients.We therefore adjusted our trial protocol, as documented in the attached file labeled Protocol No CIBI308Y014.Due to considerations related to securing trial funding and the practical limitations on the number of eligible participants, we ultimately proceeded with a single-arm trial design.We have mentioned these points in the revised Trial Design section.It is important to realize that our intention is to conduct a randomized controlled phase 3 clinical trial in the near future.When that time comes, we will gladly welcome and incorporate your valuable suggestions, ensuring the inclusion of an appropriate control group.Third, RECIST evaluation was done by the local investigators and was not confirmed centrally.Reply: Although we did not centrally confirm every image of patients from local centers, remote image assessment and verification of images were conducted at baseline and critical disease progression time points.
to the small numbers of biopsy tissue samples obtained for advanced lung squamous cell carcinoma, we could not perform PD-L1 testing on all patients.In response to the issue you raised and by Reviewer 1, we have re-analyzed the expression of PD-L1 in all tissue samples.Currently, there are PD-L1 results for 33 patients (71.7%).The remaining patients could not be tested due to the unavailability of sufficient samples.We have addressed these concerns in the revised manuscript and added new references and data.The nab-paclitaxel dose chosen (260 mg/m2 q3w) does not represent an established dose regimen for NSCLC (at least in Europe or the U.S.) and should be commented.Reply: We have added the following text to the revised discussion: Nab-paclitaxel can be administered weekly and every-3-weeks.A clinical study that compared the efficacy and safety of the weekly regimen to the 3-week regimen in NSCLC reported that in the intent-to-treat (ITT) population, the weekly regimen demonstrated certain advantages in terms of ORR and safety.However, there were no significant differences in PFS and OS between the two administration regimens.Subgroup analyses revealed that squamous cell carcinoma patients had better PFS and OS when treated with the 3-week regimen, showing significant statistical differences.Among these, the 260 mg/m² q3w dosage exhibited better tolerability (Socinski et al., 2010).Additionally, in a study of neoadjuvant chemotherapy combined with immunotherapy for NSCLC conducted in China, nab-paclitaxel at a dosage of 260 mg/m² q3w was also chosen for sq-NSCLC, and the patients tolerated it well (Qiu et al., 2022).Considering this information, the 260 mg/m² q3w dosage was selected. Socinski, M. A. et al.A dose finding study of weekly and every-3-week nab-Paclitaxel followed by carboplatin as first-line therapy in patients with advanced non-small cell lung cancer.J. Thorac.Oncol.5, 852-861 (2010). Qiu, F. et al.Two cycles versus three cycles of neoadjuvant sintilimab plus platinumdoublet chemotherapy in patients with resectable non-small-cell lung cancer (neoSCORE): A randomized, single center, two-arm phase II trial.J. Clin.Oncol.40, 8500-8500 (2022).

Table 4 | Comparison of ORR, PFS, OS and DOR between the subgroup populations with different PD-L1 expressions (n = 44)
This samples size is too small to identify the efficacy of this strategy for sq-NSCLC in stage IIIB-IV, which were composed of multiple stage.The treatment strategies of stage IIIB-C would not be unified and their prognosis is heterogeneous.Considering that the proportion of stage III patients included in this trial exceeds 20%, please carefully describe the reasons why these patients did not undergo radical surgery, neoadjuvant treatment followed by surgery, or PACIFIC regimens.For example, a portion of patients in stage IIIB who received induction immuno-chemotherapy may undergo radical resection to improve survival.
CI confidence interval, DOR duration of response, HR hazard ratio, m month, ND not detectable, NE not evaluable, ORR objective response rate, OS overall survival, PFS progression free survival, TPS tumor cell proportion score.3.